CN113215087A - Method for improving in-vitro maturation development rate of porcine oocytes by adopting agomelatine - Google Patents
Method for improving in-vitro maturation development rate of porcine oocytes by adopting agomelatine Download PDFInfo
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Abstract
The invention discloses a method for improving In-Vitro Maturation and development rate of porcine oocytes by adopting agomelatine, In particular to a method for improving In-Vitro Maturation and development rate of porcine oocytes by adopting a melatonin receptor agonist agomelatine, which obviously improves the amplification of porcine oocytes, In Vitro Maturation (IVM) of oocytes, the expression of genes related to oocyte development and the development capability of parthenogenetic embryos.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to the technical field of oocyte in-vitro maturation culture, relates to a method for improving the in-vitro maturation development rate of porcine oocytes by adopting agomelatine, and particularly relates to a method for improving the in-vitro maturation development rate of porcine oocytes by adopting a melatonin receptor agonist agomelatine.
[ background of the invention ]
The pig is similar to human beings in the aspects of anatomy, physiology, genetic background, disease characteristics and the like, and is considered as a first choice animal of human xenogeneic organ sources, so the pig is an important model animal in biomedical research. The effective combination of in vitro production technology and gene editing technology not only promotes the development and application of embryo engineering, but also drives the development of the medical field. In Vitro embryo Production (IVP) requires large quantities of high quality oocytes. The in vitro culture and maturation of oocytes are the basis and key link of embryo biotechnology. At present, embryo technology has greatly promoted the processes of animal husbandry and medicine, such as breed improvement, transgenic disease-resistant breed cultivation, preparation of human disease models, production of animal bioreactor medicinal protein and research of development mechanism, etc.
The somatic cell cloning and disease model preparation of pigs need a large amount of oocytes with development potential, but the in vitro maturation rate of the prior porcine oocytes is not ideal.
Currently, porcine oocytes are cultured in vitro culture media such as the North Carolina State University (NCSU23/NCSU37), PZM series (PZM-3/PZM-4/PZM-5) and TCM-199, of which TCM-199 is the most successful culture medium recognized to substantially meet the in vitro developmental metabolism and nutritional needs of porcine oocytes.
Compared with the in vivo environment, in the in vitro culture process of the oocyte and the embryo, the cell is in a relatively high oxygen environment, and oxidative stress is often caused by insufficient antioxidant defense, so that a large amount of Reactive Oxygen Species (ROS) are accumulated in the cell in the development process, and the in vitro development rate of the oocyte and the embryo is reduced.
Agomelatine (Agomelatine) is an MT1 and MT2 receptor agonist, has a longer half-life than melatonin, and has a higher affinity than melatonin. At present, no research is available for adding the melatonin receptor agonist agomelatine in the in vitro maturation process of porcine oocytes. Therefore, the research on whether the agomelatine can obviously improve the antioxidant capacity and reduce the apoptosis rate of the oocyte or not has important significance on improving the in vitro maturation of the oocyte and the in vitro development capacity of the embryo.
[ summary of the invention ]
The invention provides a method for improving the In Vitro Maturation and development rate of porcine oocytes by adopting agomelatine, In particular a method for improving the In Vitro Maturation and development rate of porcine oocytes by adopting a melatonin receptor agonist agomelatine, which is used for remarkably improving the amplification of porcine oocytes, In Vitro Maturation (IVM) of oocytes, expression of genes related to oocyte development and the embryo development capability of parthenogenesis.
The method for improving the in vitro maturation and development rate of the porcine oocytes by adopting agomelatine comprises the following steps:
1) preparing maturation culture solution of oocyte:
a culture solution: 80% by volume TCM199, 10% by volume porcine follicular fluid, 10% by volume fetal bovine serum, 0.1g/L cysteine, 0.075g/L penicillin, 0.05g/L streptomycin, 10ng/mL insulin-like growth factor, 50ng/mL epidermal growth factor, 2.2g/L NaHCO310IU/mL pregnant mare serum gonadotropin, 10IU/mL human chorionic gonadotropin, from 10-11To 10-8Agomelatine in mol/L concentration;
b, culture solution: 80% volume TCM199, 10% volume porcine follicular fluid, 10% volume fetal bovine serum, 0.1g/L cysteine, 0.075g/L penicillin, 0.05g/L streptomycin, 10ng/mL insulin-like growth factor, 50ng/mL epidermal growth factor, 2.2g/L NaHCO3From 10-11To 10-8Agomelatine in mol/L concentration;
2) preparing an electric activation liquid: 0.3mmol/L mannitol, 0.1mmol/L CaCl2·H2O、0.1mmol/L MgSO4·7H2O、0.5mmol/L Hepes、0.01％(w/v)PVA；
3) Preparing embryo culture solution: adopts PZM3 embryo culture solution, which comprises the following components: 6.31g/L NaCl, 0.74g/L KCl, 0.047g/L KH2PO4、2.1g/L NaHCO30.022g/L of sodium pyruvate, 0.62g/L of calcium lactate, 0.15g/L L-glutamine, 0.55g/L of hypotaurine, 20ml/L of essential amino acid, 10ml/L of nonessential amino acid, 0.065g/L of penicillin, 0.05g/L of streptomycin and 4g/L of BSA;
4) preparation of pig follicular fluid: drawing 3-8mm transparent follicles from pig ovaries to obtain follicular fluid, centrifuging the fluid, collecting supernatant, and filtering with 0.22 μm filter to remove impurities to obtain required follicular fluid;
5) and (3) culturing the oocyte: washing pig ovary with 75% ethanol for 15s, washing with physiological saline containing penicillin and streptomycin at 37 deg.C for 3 times, gently extracting 2-6mm clear follicle with 10mL syringe equipped with 12-gauge needle, and placing into test tube in 38.5 deg.C constant temperature rack; selecting Cumulus Oocyte Complexes (COCs) which contain 3 layers or more than 3 layers of particles, are coated by cumulus cells and have good refractivity under a stereoscopic microscope, transferring the oocytes into the culture solution A in the step 1), culturing for 20-22h, and then transferring to the culture solution B in the step 1) to culture for 20-22 h;
6) parthenogenetic (PA) embryo production: cleaning the fusion tank with 15min balanced activating liquid for 3 times, cleaning the oocyte in the activating liquid for 3 times, transferring to the middle of the fusion tank covered with the activating liquid, activating with 1.00kV/cm and 80 μ s DC pulse for 3 times, cleaning the electrically activated oocyte in PZM3 embryo culture liquid for 3 times, transferring to the microdrops of the embryo culture liquid, and continuously culturingCultivating; the above-mentioned continuous culture condition is 5% CO2And dyeing the blastocyst cultured for 144h in Hoechst33342 for 15min at the saturation humidity of 39 ℃, dripping 1-2 drops of glycerol for tabletting and sealing, uniformly spreading the blastocyst on a glass slide, and counting cells under the fluorescent irradiation.
In the invention:
from 10 as described in step 1)-11To 10-8Agomelatine in a mol/L concentration of 10 each-11、10-10、10-9、10-8Agomelatine in mol/L.
Further, the concentration of the agomelatine in the step 1) is 10-10mol/L。
Centrifuging in the step 4), wherein the rotating speed is 3500r/min and the time is 15 min.
The Hoechst33342 of the step 6) is used in an amount of 10 mu g/mL.
Compared with the prior art, the invention has the following advantages:
1. according to the method for improving the in-vitro maturation and development rate of the porcine oocytes by adopting agomelatine, the melatonin receptor agonist agomelatine is added into the maturation culture solution of the oocytes, so that the blastocyst development capability and the blastocyst quality can be improved by promoting the cytoplasm maturation. Lower concentrations of agomelatine (10) melatonin receptor agonist in promoting oocyte maturation in vitro-10mol/L) of melatonin (10) at a specific optimum concentration-8mol/L) is more excellent.
2. According to the method for improving the in vitro maturation and development rate of the porcine oocytes by using the agomelatine, the agomelatine is added to enable cumulus cells to be better diffused; the expression levels of cell maturation related genes BMP15 and GDF9 and antioxidant related gene CAT in the oocyte are improved, and the expression levels of apoptosis promoting genes Caspase6 and P53 are reduced, so that the quality of the porcine oocyte and the in vitro developmental capacity of PA embryo are improved.
[ description of the drawings ]
FIG. 1 shows the optimum concentration 10 in example 2 of the present invention-10mol/L agomelatine-treated group, comparative example 1 blank control group, and comparative example 2 melatonin treatmentSet of graphs illustrating the effects of expression of the oocyte development-associated gene BMP15 (10 from left to right, respectively)-10Relative expression amounts of BMP15 genes in mol/L agomelatine-treated group, blank control group, and melatonin group).
FIG. 2 shows the optimum concentration 10 in example 2 of the present invention-10Graph of the effect of mol/L agomelatine-treated group, control blank of comparative example 1, and melatonin-treated group of comparative example 2 on the expression of the oocyte development-related gene GDF9 (10 from left to right, respectively)-10Relative expression amounts of GDF9 genes in mol/L agomelatine-treated group, blank control group, and melatonin group).
FIG. 3 shows the optimum concentration 10 in example 2 of the present invention-10Graph of the effect of mol/L agomelatine-treated group, control blank of comparative example 1, and melatonin-treated group of comparative example 2 on the expression of the oocyte development-related gene Caspase6 (10 from left to right, respectively)-10Relative expression amounts of Caspase6 genes in mol/L agomelatine-treated group, blank control group, and melatonin group).
FIG. 4 shows the optimum concentration 10 in example 2 of the present invention-10Graph of the effect of mol/L agomelatine-treated group, control blank of comparative example 1, and melatonin-treated group of comparative example 2 on the expression of oocyte development-related gene P53 (10 from left to right, respectively)-10Relative expression amounts of P53 genes in mol/L agomelatine-treated group, blank control group, and melatonin group).
FIG. 5 shows the optimum concentration 10 in example 2 of the present invention-10Graph of the effect of mol/L agomelatine-treated group, control blank of comparative example 1, and melatonin-treated group of comparative example 2 on the expression of the gene CAT involved in oocyte development (10 from left to right, respectively)-10Relative expression amounts of CAT genes in the mol/L agomelatine-treated group, the blank control group and the melatonin group).
[ detailed description ] embodiments
The following examples are provided to further illustrate the embodiments of the present invention.
Example 1:
the method for improving the in vitro maturation and development rate of the porcine oocytes by adopting agomelatine comprises the following steps:
1) preparing maturation culture solution of oocyte:
a culture solution: 80% by volume TCM199, 10% by volume porcine follicular fluid, 10% by volume fetal bovine serum, 0.1g/L cysteine, 0.075g/L penicillin, 0.05g/L streptomycin, 10ng/mL insulin-like growth factor, 50ng/mL epidermal growth factor, 2.2g/L NaHCO310IU/mL pregnant mare serum gonadotropin, 10IU/mL human chorionic gonadotropin, 10IU/mL- 11Agomelatine in mol/L concentration;
b, culture solution: 80% by volume TCM199, 10% by volume porcine follicular fluid, 10% by volume fetal bovine serum, 0.1g/L cysteine, 0.075g/L penicillin, 0.05g/L streptomycin, 10ng/mL insulin-like growth factor, 50ng/mL epidermal growth factor, 2.2g/L NaHCO3、10-11Agomelatine in mol/L concentration;
2) preparing an electric activation liquid: 0.3mmol/L mannitol, 0.1mmol/L CaCl2.H2O、0.1mmol/L MgSO4·7H2O、0.5mmol/L Hepes、0.01％(w/v)PVA；
3) Preparing embryo culture solution: adopts PZM3 embryo culture solution, which comprises the following components: 6.31g/L NaCl, 0.74g/L KCl, 0.047g/L KH2PO4, 2.1g/L NaHCO30.022g/L of sodium pyruvate, 0.62g/L of calcium lactate, 0.15g/L L-glutamine, 0.55g/L of hypotaurine, 20ml/L of essential amino acid, 10ml/L of nonessential amino acid, 0.065g/L of penicillin, 0.05g/L of streptomycin and 4g/L of BSA;
4) preparation of pig follicular fluid: drawing 3-8mm transparent follicle from pig ovary to obtain follicular fluid, centrifuging at 3500r/min for 15min, collecting supernatant, and filtering with 0.22 μm filter to remove impurities to obtain required follicular fluid;
5) and (3) culturing the oocyte: washing pig ovary with 75% ethanol for 15s, washing with physiological saline containing penicillin and streptomycin at 37 deg.C for 3 times, gently extracting 2-6mm clear follicle with 10mL syringe equipped with 12-gauge needle, and placing into test tube in 38.5 deg.C constant temperature rack; selecting Cumulus Oocyte Complexes (COCs) which contain 3 layers or more than 3 layers of particles, are coated by cumulus cells and have good refractivity under a stereoscopic microscope, transferring the oocytes into the culture solution A of the step 1) in the example 1, culturing for 20-22h, and then changing to the culture solution B of the step 1) in the example 1 to culturing for 20-22 h;
6) parthenogenetic (PA) embryo production: cleaning the fusion tank for 3 times by using an activation solution which is balanced for 15min by adopting an electric activation method, cleaning the oocyte in the activation solution for 3 times, moving the oocyte to the middle of an electric fusion tank which is covered with the electric activation solution, activating the oocyte for 3 times by using direct current pulse of 1.00kV/cm and 80 mu s, placing the electrically activated oocyte in PZM3 embryo culture solution for 3 times, and moving the oocyte into microdrops of the embryo culture solution for continuous culture; the above-mentioned continuous culture condition is 5% CO2And dyeing the blastocyst cultured for 168h in Hoechst33342 of 10 mu g/mL for 15min at the saturation humidity of 39 ℃, dripping 1-2 drops of glycerol for tabletting and sealing, uniformly spreading the glycerol on a glass slide, and counting cells under the fluorescent irradiation.
Example 2:
method for improving in vitro maturation and development rate of porcine oocytes by using agomelatine, compared with example 1, the dosage of agomelatine in step 1) is 1 × 10-10mol/L, otherwise the same as example 1.
Example 3:
method for improving in vitro maturation and development rate of porcine oocytes by using agomelatine, compared with example 1, the dosage of agomelatine in step 1) is 1 × 10-9mol/L, otherwise the same as example 1.
Example 4:
method for improving in vitro maturation and development rate of porcine oocytes by using agomelatine, compared with example 1, the dosage of agomelatine in step 1) is 1 × 10-8mol/L, otherwise the same as example 1.
Comparative example 1:
compared with the example 1, the dosage of the agomelatine in the step 1) is 0mol/L, and the other steps are the same as the example 1.
Comparative example 2:
compared with example 1, in step 1), agomelatine was replaced by melatonin, and the dosage of melatonin was 10-8mol/L, wherein:
a culture solution: 80% by volume TCM199, 10% by volume porcine follicular fluid, 10% by volume fetal bovine serum, 0.1g/L cysteine, 0.075g/L penicillin, 0.05g/L streptomycin, 10ng/mL insulin-like growth factor, 50ng/mL epidermal growth factor, 2.2g/L NaHCO310IU/mL pregnant mare serum gonadotropin, 10IU/mL human chorionic gonadotropin, 10IU/mL- 8mol/L melatonin;
b, culture solution: 80% by volume TCM199, 10% by volume porcine follicular fluid, 10% by volume fetal bovine serum, 0.1g/L cysteine, 0.075g/L penicillin, 0.05g/L streptomycin, 10ng/mL insulin-like growth factor, 50ng/mL epidermal growth factor, 2.2g/L NaHCO3、10-8mol/L melatonin;
the rest is the same as example 1.
Experimental example:
1. cumulus expansion degree experiment of oocytes:
the criteria for the extent of cumulus expansion of the oocytes are:
grade 0 indicates that cumulus cells of the oocytes do not spread;
grade 1+ indicates that cumulus cells of the oocytes only diffuse in the outermost layer, and the outer layer of granulosa cells can see flash luminescence;
grade 3+ indicates that cumulus cells of the oocytes are all diffused except the radioactive crown; grade 4+ indicates full spread of cumulus cells of the oocytes.
As a result:
the treated groups (examples 1 to 4), the control group (comparative example 1) and the melatonin treated group (comparative example 2) were observed for cumulus expansion, data were recorded, and cumulus expansion index (cumulus expansion index was calculated by the following algorithm: CEI ═ 0+1 grade number of cells × 1+2 grade number of cells × 2+3 grade number of cells × 3+4 grade number of cells × 4) ÷ total number of cells) was calculated, as shown in table 1:
TABLE 1 Effect of different concentrations of Agomelatine-treated group, placebo group and melatonin-treated group on pig cumulus cell spreading index
Note: the data in the same column are marked with different letters to represent that the data in the same column are different remarkably (P <0.05), and no letters or the same letters are included to represent that the data are not different remarkably (P > 0.05).
From Table 1, it can be seen that 10 is added-10The diffusing index of the cumulus cells of the mol/L agomelatine-treated oocytes is obviously higher than that of other concentration-treated groups, control groups and melatonin-treated groups.
2. Comparing the oocyte apoptosis rate and the first polar body discharge rate of the agomelatine-treated group, the control group and the melatonin-treated group at different concentrations, the results are shown in table 2:
TABLE 2 Effect of Agomelatine-treated group, placebo group and melatonin-treated group at different concentrations on the apoptosis rate and first polar body discharge rate of oocytes
Note: the data in the same column are marked with different letters to represent that the data in the same column are different remarkably (P <0.05), and no letters or the same letters are included to represent that the data are not different remarkably (P > 0.05).
From Table 2, it can be seen that 10 is added-10The apoptosis rate of the egg mother cells treated by mol/L agomelatine is obviously lower than that of other concentration treatment groups, control groups and melatonin treatment groups; for the first pole body discharge rate 10-10The mol/L agomelatine treatment group is obviously higher than the other concentration treatment group, the control group and the melatonin treatment group.
3. And detecting the mRNA level expression of the porcine oocyte related genes of the optimal concentration treatment group, the control group and the melatonin treatment group by using a qRT-PCR technology. Mainly detects oocyte maturation related genes BMP15 and GDF 9; the apoptosis-promoting genes Caspase6 and P53; antioxidation-related gene CAT (see tables 3 and 4 and FIG. 1 for details).
TABLE 3 real-time fluorescent quantitative PCR primer sequences
TABLE 4 optimum concentration (10)-10mol/L) influence of Agomelatine treatment group, blank control group and melatonin treatment group on expression of pig oocyte development related genes
Note: the data in the same column marked with different letters represents that the data in the same column has obvious difference (P <0.05), no letters or the same letters represent that the data in the same column has no obvious difference (P >0.05)
Fig. 1-5 show the expression influence of the genes related to oocyte development in the agomelatine treatment group, the blank control group and the melatonin treatment group with the optimal concentration, and the data in the same column are marked with different letters to show that the data difference is significant and is less than 0.05.
From fig. 1-2, it can be seen that the expression levels of the oocyte maturation associated genes BMP15 and GDF9 in the agomelatine treatment group with the optimal concentration are significantly higher than those in the control group and the melatonin treatment group, and the melatonin treatment group is significantly higher than that in the control group, which indicates that the optimal concentration agomelatine treatment can promote the in vitro development and maturation of the oocytes; 3-4, the expression levels of the apoptosis-promoting genes Caspase6 and P53 are obviously lower than those of a control group and a melatonin treatment group, and the control group is obviously higher than that of the melatonin treatment group, so that the situation that the apoptosis rate of the in vitro development of the oocyte can be reduced by the treatment of the agomelatine with the optimal concentration is shown; from fig. 5, it can be seen that the expression level of the antioxidant related gene CAT is significantly higher than that of the control group and the melatonin treatment group, which indicates that the antioxidant capacity of the oocytes in vitro development can be improved by the treatment of agomelatine with the optimal concentration.
TABLE 5 Effect of treatment with different concentrations of Agomelatine on the embryonic development Capacity of PA
From Table 5, it can be seen that 10-11mol/L agomelatine, 10-10Agomelatine and 10 mol/L-8The cleavage rate of the PA embryo in the mol/L melatonin treatment group is higher and is obviously higher than that of other concentration treatment groups and control groups; in terms of blastocyst Rate and blastocyst cell number, 10-10The highest of the mol/L agomelatine treatment group is obviously higher than the blastocyst rate and the total number of blastocysts of other concentration treatment groups, a control group and a melatonin treatment group.
Analysis and discussion:
agomelatine is a melatonin receptor agonist, has strong affinity and intrinsic activity to MT1 and MT2 receptors, and can exert the maximum effect through receptor excitation. Early studies show that melatonin can promote oocyte maturation and embryo development of mammals such as mice, sheep, cattle and the like. Melatonin is added in the in-vitro maturation process of the porcine oocytes, the GSH level is improved although the maturation rate of the oocytes is not improved, and the blastocyst rate and the number of blastocysts of the pigs are increased. In the invention, agomelatine is used for replacing melatonin, and better effects can be obtained by promoting the maturation of cytoplasm and improving the blastocyst development capability and the quality of blastocysts.
In addition, previous studies have also found that 10 is added during in vitro maturation of porcine oocytes-8mol/L melatonin, which enhances blastocyst development, and low concentrations of melatonin receptor agonist 10 in this study-10mol/L agomelatine and high-concentration 10-8The results of the mol/L melatonin treatment are very similar, which is likely related to the higher avidity of agomelatine compared to melatonin.
The improvement of the expression levels of cell maturation related genes BMP15 and GDF9 and antioxidant related gene CAT possibly improves the culture environment of the oocyte in the oocyte maturation culture solution and improves the maturation of the oocyte cytoplasm; the expression levels of the apoptosis-promoting genes Caspase6 and P53 are reduced, and the apoptosis of the oocyte is reduced.
And (4) conclusion:
during in vitro maturation of porcine oocytes, 10 is added-10The agomelatine/L enables cumulus cells to be better diffused; the expression levels of cell maturation related genes BMP15 and GDF9 and antioxidant related gene CAT in the oocyte are improved, and the expression levels of apoptosis promoting genes Caspase6 and P53 are reduced, so that the quality of the porcine oocyte and the in vitro developmental capacity of PA embryo are improved.
The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the claims of the present invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the claims of the present invention.
Claims (5)
1. The method for improving the in vitro maturation and development rate of the porcine oocytes by adopting agomelatine is characterized by comprising the following steps: the method comprises the following steps:
1) preparing maturation culture solution of oocyte:
a culture solution: 80% by volume TCM199, 10% by volume porcine follicular fluid, 10% by volume fetal bovine serum, 0.1g/L cysteine, 0.075g/L penicillin, 0.05g/L streptomycin, 10ng/mL insulin-like growth factor, 50ng/mL epidermal growth factor, 2.2g/L NaHCO310IU/mL pregnant mare serum gonadotropin, 10IU/mL human chorionic gonadotropin, from 10-11To 10-8Agomelatine in mol/L concentration;
b, culture solution: 80% by volume TCM199, 10% by volume porcine follicular fluid, 10% by volume fetal bovine serum, 0.1g/L cysteine, 0.075g/L penicillin, 0.05g/L streptomycin, 10ng/mL insulin-like growth factor, 50ng/mL epidermal growth factor, 2.2g/L NaHCO3From 10-11To 10-8Agomelatine in mol/L concentration;
2) preparing an electric activation liquid: 0.3mmol/L mannitol, 0.1mmol/L CaCl2·H2O、0.1mmol/L MgSO4·7H2O、0.5mmol/L Hepes、0.01％w/v PVA；
3) Preparing embryo culture solution: adopts PZM3 embryo culture solution, which comprises the following components: 6.31g/L NaCl, 0.74g/L KCl, 0.047g/L KH2PO4、2.1g/L NaHCO30.022g/L of sodium pyruvate, 0.62g/L of calcium lactate, 0.15g/L L-glutamine, 0.55g/L of hypotaurine, 20ml/L of essential amino acid, 10ml/L of nonessential amino acid, 0.065g/L of penicillin, 0.05g/L of streptomycin and 4g/L of BSA;
4) preparation of pig follicular fluid: drawing 3-8mm transparent follicles from pig ovaries to obtain follicular fluid, centrifuging the fluid, collecting supernatant, and filtering with 0.22 μm filter to remove impurities to obtain required follicular fluid;
5) and (3) culturing the oocyte: washing pig ovary with 75% ethanol for 15s, washing with physiological saline containing penicillin and streptomycin at 37 deg.C for 3 times, gently extracting 2-6mm clear follicle with 10mL syringe equipped with 12-gauge needle, and placing into test tube in 38.5 deg.C constant temperature rack; selecting a cumulus oocyte complex which contains 3 layers or more than 3 layers of particles, is coated by cumulus cells and has good refractivity under a stereoscopic microscope, transferring the oocyte into the culture solution A in the step 1), culturing for 20-22h, and then transferring to the culture solution B in the step 1) to culture for 20-22 h;
6) parthenogenetic embryo production: cleaning the fusion tank for 3 times by using an activation solution which is balanced for 15min by adopting an electric activation method, cleaning the oocyte in the activation solution for 3 times, moving the oocyte to the middle of the fusion tank covered with the electric activation solution, activating the oocyte for 3 times by using direct current pulse of 1.00kV/cm and 80 mu s, placing the electrically activated oocyte in PZM3 embryo culture solution for 3 times, and moving the oocyte into microdrops of the embryo culture solution for continuous culture; the above-mentioned continuous culture condition is 5% CO2And dyeing the blastocyst cultured for 144h in Hoechst33342 for 15min at the saturation humidity of 39 ℃, dripping 1-2 drops of glycerol for tabletting and sealing, uniformly spreading the blastocyst on a glass slide, and counting cells under the fluorescent irradiation.
2. The method for improving the in vitro maturation and development rate of porcine oocytes using agomelatine according to claim 1, wherein: step 1) theFrom 10 to-11To 10-8Agomelatine in a mol/L concentration of 10 each-11、10-10、10-9、10-8Agomelatine in mol/L.
3. The method for improving the in vitro maturation and development rate of porcine oocytes using agomelatine according to claim 2, wherein: the concentration of the agomelatine in the step 1) is 10-10mol/L。
4. The method for improving the in vitro maturation and development rate of porcine oocytes using agomelatine according to claim 1, wherein: centrifuging in the step 4), wherein the rotating speed is 3500r/min and the time is 15 min.
5. The method for improving the in vitro maturation and development rate of porcine oocytes using agomelatine according to claim 1, wherein: the Hoechst33342 of the step 6) is used in an amount of 10 mu g/mL.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113174366A (en) * | 2021-03-29 | 2021-07-27 | 广西壮族自治区畜牧研究所 | In-vitro maturation system of porcine oocyte containing butylbenzohydroxy acid and application thereof |
| CN117143809A (en) * | 2023-10-30 | 2023-12-01 | 云南省畜牧兽医科学院 | Method for improving in-vitro maturation quality of porcine oocytes |
| CN117143809B (en) * | 2023-10-30 | 2023-12-26 | 云南省畜牧兽医科学院 | Method for improving in-vitro maturation quality of porcine oocytes |
| CN117511854A (en) * | 2024-01-05 | 2024-02-06 | 金宝医学科技(深圳)有限公司 | Immature oocyte culture solution and preparation method thereof |
| CN117511854B (en) * | 2024-01-05 | 2024-03-19 | 金宝医学科技(深圳)有限公司 | Immature oocyte culture solution and preparation method thereof |
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